ABSTRACT The increase in obesity worldwide has led to rising health care costs and the number of chronically ill people suffering from obesity-related disorders such as type 2 diabetes. The overarching goal of this research proposal is to better understand brown fat activation and to develop new therapeutic manipulations targeting brown fat to treat metabolic disease such as type 2 diabetes. This work will test the hypothesis that the secreted protein Slit2 is involved in adipose thermogenesis and if administration of the protein could be a safe and effective treatment for diabetes. We have previously utilized unbiased quantitative proteomics to identify novel secreted proteins involved in browning. Using this method, I have identified new mechanisms by which thermogenesis can be activated in adipose tissue. One of the candidates from this approach identified Slit2 as a secreted factor from thermogenic adipocytes. Slit2 had previously been described for its function in brain, and were not believed to be circulating in blood. In addition, I have found a C-terminal cleavage fragment of Slit2, Slit2-C, which has no known peripheral function and acts through an unknown cell-surface receptor. My preliminary data demonstrate that increasing Slit2-C circulating levels in obese, insulin resistant mice improves whole body glucose homeostasis and energy expenditure. These mice show increased oxygen consumption in the brown fat tissue as well as activation of a thermogenic transcriptional program. At least in part, Slit2-C acts through activation of the canonical PKA pathway. In the mentored phase of the award, Aim 1 will test the hypothesis that Slit2 is required for regulating adipose tissue thermogenesis in vivo by performing metabolic characterization of the adipocyte-specific knockout of Slit2. In aim 2 of the mentored phase, I will test the hypothesis that administration of Slit2-C recombinant protein to diabetic rodents will improve diet-induced insulin resistance. Aim 3 will be conducted in the independent phase and will use multiple approaches to determine the functional receptor and signaling pathways for Slit2-C with further potential for clinical translation. I will use a combination of animal physiology and genetics, biochemical protein purification, and mass spectrometry techniques to address the questions in the proposal. If successful, I anticipate that the findings in this proposal has the potential to contribute with new treatments for type-2 diabetes. My current and long-term career objectives are to identify pathways involved in adipose tissue metabolism and to develop new protein therapeutics that regulates glucose homeostasis and has the possibility to improve diabetes. I have a longstanding interest in studying ligand-receptor interactions, macromolecular uptake and mechanisms of intracellular signaling in tumor development. The findings in this proposal are directly building upon my discovery in my postdoctoral work and would be completed in the defined award period. Therefore, study of secreted factor Slit2-C as a new protein therapy for diabetes is a logical extension of my research. My career trajectory after the mentored phase of the K99 is to become an assistant professor at a leading academic research institute. Dr. Bruce Spiegelman, a well-recognized leader in the field of brown fat and diabetes will mentor my scientific and career development. Dr. Spiegelman has successfully trained numerous postdoctoral fellows now holding faculty positions in academic institutions. The Spiegelman laboratory and Harvard Medical School research community provide an ideal setting for training future independent investigators. My plan for career activities includes a continous progress evaluation with my mentor, training in biochemistry and protein therapies, supervision and leadership training, educational coursework, and mentored job search. These career activities will be imperative in the preparation to establish my own research laboratory. In the independent phase, I plan to leverage my biochemical skills and development of new protein biologics and signaling pathways to scientifically separate myself from my mentor. My long-term scientific goal is to establish myself as an independent researcher to drive the field of metabolism and diabetes forward. My focus will be to study biological pathways of circulating hormones and to further study adrenergic receptor-independent pathways of thermogenesis activation in the context of obesity and diabetes. Together with the outstanding resources at Harvard Medical School, this will maximize my potential to successfully transition to independence. The NIH Pathway to Independence Award will be essential for my transition because it will enable me to gain additional training in mouse models of obesity and protein therapies as well as improving my writing and communication skills required for a successful transition.